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Phase Contrast and Differential Interference Contrast Microscopy01:26

Phase Contrast and Differential Interference Contrast Microscopy

Phase-Contrast Microscopes
In-phase-contrast microscopes, interference between light directly passing through a cell and light refracted by cellular components is used to create high-contrast, high-resolution images without staining. It is the oldest and simplest type of microscope that creates an image by altering the wavelengths of light rays passing through the specimen. Altered wavelength paths are created using an annular stop in the condenser. The annular stop produces a hollow cone of...
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A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors
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Phase optimisation for structured illumination microscopy.

Kai Wicker1, Ondrej Mandula, Gerrit Best

  • 1Institute of Physical Chemistry, Abbe Center of Photonics, Friedrich-Schiller-University Jena, Jena, Germany. kai.wicker@gmail.com

Optics Express
|February 8, 2013
PubMed
Summary
This summary is machine-generated.

Structured illumination microscopy (SIM) achieves super-resolution fluorescence imaging. A new algorithm precisely retrieves illumination pattern phases from acquired data, enabling artifact-free super-resolution images even with fine patterns.

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Area of Science:

  • Optical microscopy
  • Fluorescence imaging
  • Super-resolution techniques

Background:

  • Structured illumination microscopy (SIM) enables super-resolution fluorescence imaging by illuminating samples with patterned light.
  • Accurate knowledge of illumination pattern phases is critical for artifact-free image reconstruction in SIM.
  • Experimental limitations can prevent achieving the required phase precision, necessitating post-acquisition phase retrieval.

Purpose of the Study:

  • To develop a fast and robust algorithm for accurate a posteriori determination of illumination pattern phases in SIM.
  • To enable artifact-free super-resolution image reconstruction when experimental phase precision is insufficient.
  • To provide a reliable method for phase optimization, even for very fine illumination patterns undetectable by other means.

Main Methods:

  • Development of an iterative algorithm based on cross-correlations for phase retrieval.
  • Application of the algorithm to simulated 2D sample data and experimental 3D sample data.
  • Comparative analysis of the developed method against a previously published phase retrieval approach.

Main Results:

  • The algorithm iteratively determines illumination pattern phases with high precision, typically below λ/100.
  • The cross-correlation-based method successfully optimizes pattern phases for fine patterns, where other methods fail.
  • Performance analysis using both simulated and experimental data validates the algorithm's robustness and accuracy.

Conclusions:

  • The presented algorithm offers a fast, robust, and highly precise solution for retrieving illumination pattern phases in structured illumination microscopy.
  • This method significantly improves the quality of super-resolution images by enabling artifact-free reconstruction even under challenging experimental conditions.
  • The algorithm's ability to handle fine patterns makes it a valuable tool for advanced fluorescence imaging applications.